RESUMO
Classical swine fever virus (CSFV) is a single-stranded RNA flavivirus that can cause serious diseases in porcine species, including symptoms of infarction, systemic hemorrhage, high fever, or depression. Viperin is an important interferon-inducible antiviral gene that has been shown to inhibit CSFV, but the exact mechanisms by which it is able to do so remain poorly characterized. In the present study, we determined that CSFV infection led to viperin upregulation in PK-15 cells (porcine kidney cell). When viperin was overexpressed in these cells, this markedly attenuated CSFV replication, with clear reductions in viral copy number after 12 to 48 hours postinfection. Immunofluorescence microscopy revealed that the viral NS5A protein colocalized with viperin in infected cells, and this was confirmed via confocal laser scanning microscopy using labeled versions of these proteins, and by co-immunoprecipitation which confirmed that NS5A directly interacts with viperin. When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N-terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction. Together, our in vitro results highlight a potential mechanism whereby viperin is able to inhibit CSFV replication. These results have the potential to assist future efforts to prevent or treat systemic CSFV-induced disease, and may also offer more general insights into the antiviral role of viperin in innate immunity.
Assuntos
Vírus da Febre Suína Clássica/fisiologia , Peste Suína Clássica/imunologia , Proteínas/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Peste Suína Clássica/genética , Vírus da Febre Suína Clássica/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunoprecipitação , Interferons/fisiologia , Domínios e Motivos de Interação entre Proteínas , Proteínas/genética , Transdução de Sinais , Suínos , Proteínas não Estruturais Virais/genéticaRESUMO
OBJECTIVES: To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T. RESULTS: MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells. CONCLUSIONS: LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.
Assuntos
Células Epiteliais/citologia , Lipopolissacarídeos/farmacologia , NF-kappa B/genética , NF-kappa B/metabolismo , Pirrolidinas/farmacologia , Tiocarbamatos/farmacologia , Acidose/genética , Acidose/metabolismo , Acidose/patologia , Animais , Apoptose/efeitos dos fármacos , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacosRESUMO
To explore the role of IRF3/IRF7 during inflammatory responses, we investigated the effects of swine IRF3/IRF7 on TLR4 signaling pathway and inflammatory factors expression in porcine kidney epithelial PK15 cell lines. We successfully constructed eukaryotic vectors PB-IRF3 and PB-IRF7, transfected these vectors into PK15 cells and observed GFP under a fluorescence microscope. In addition, RT-PCR was also used to detect transfection efficiency. We found that IRF3/IRF7 was efficiently overexpressed in PK15 cells. Moreover, we evaluated the effects of IRF3/IRF7 on the TLR4 signaling pathway and inflammatory factors by RT-PCR. Transfected cells were treated with lipopolysaccharide (LPS) alone, or in combination with a TBK1 inhibitor (LiCl). We revealed that IRF3/IRF7 enhanced IFNα production, and decreased IL-6 mRNA expression. Blocking the TBK1 pathway, inhibited the changes in IFNα, but not IL-6 mRNA. This illustrated that IRF3/IRF7 enhanced IFNα production through TLR4/TBK1 signaling pathway and played an anti-inflammatory role, while IRF3/IRF7 decreased IL-6 expression independent of the TBK1 pathway. Trends in MyD88, TRAF6, TBK1 and NFκB mRNA variation were similar in all treatments. LPS increased MyD88, TRAF6, TBK1 and NFκB mRNA abundance in PBR3/PBR7 and PBv cells, while LiCl blocked the LPS-mediated effects. The levels of these four factors in PBR3/PBR7 cells were higher than those in PBv. These results demonstrated that IRF3/IRF7 regulated the inflammatory response through the TLR4 signaling pathway. Overexpression of swine IRF3/IRF7 in PK15 cells induced type I interferons production, and attenuated inflammatory responses through TLR4 signaling pathway.
RESUMO
IFITM3 is involved in cell adhesion, apoptosis, immune, and antivirus activity. Furthermore, IFITM3 gene has been considered as a preferential marker for inflammatory diseases, and positive correlation to pathological grades. Therefore, we assumed that IFITM3 was regulated by different signal pathways. To better understand IFITM3 function in inflammatory response, we cloned swine IFITM3 gene, and detected IFITM3 distribution in tissues, as well as characterized this gene. Results indicated that the length of swine IFITM3 gene was 438 bp, encoding 145 amino acids. IFITM3 gene expression abundance was higher in spleen and lungs. Moreover, we next constructed the eukaryotic expression vector PBIFM3 and transfected into PK15 cells, finally obtained swine IFITM3 gene stable expression cell line. Meanwhile, we explored the effects of LPS on swine IFITM3 expression. Results showed that LPS increased IFITM3 mRNA abundance and exhibited time-dependent effect for LPS treatment. To further demonstrate the mechanism that IFITM3 regulated type I IFNs production, we also detected the important molecules expression of TLR4 signaling pathway. In transfected and non-transfected IFITM3 PK15 cells, LPS exacerbated the relative expression of TLR4-NFκB signaling molecules. However, the IFITM3 overexpression suppressed the inflammatory development of PK15 cells. In conclusion, these data indicated that the overexpression of swine IFITM3 could decrease the inflammatory response through TLR4 signaling pathway, and participate in type I interferon production. These findings may lead to an improved understanding of the biological function of IFITM3 in inflammation.